The physicochemical properties of the synthesized nanoparticles were characterized and compared, including their size, surface charge, and morphology. In addition, in vitro studies were also performed using two pancreatic cancer cell lines, assessing the cell viability of the PBDFDTBO-included PEGylated nanoparticles formulations. Moreover, in vivo studies were also conducted, using subcutaneous murine cancer models to investigate the polymeric nanoparticles’ circulation time, tumor accumulation, and preferred organ biodistribution. The overall results demonstrated that even with the same PEGylated surface, the hydrophobic composition anchored on the encapsulated PBDFDTBO core strongly affected the biodistribution and tumor accumulation of the nanoparticles, to a degree possibly determined by the hydrophobic interactions between the hydrophobic segment of amphiphilic polymers (DSPE or PCL moiety) and the enwrapped PBDFDTBO. Both PEGylated nanoparticles were compared to obtain an optimized coating strategy for a desired biological feature in pancreatic cancer delivery.In this study, a chemically synthetic polymer, benzo[1,2- Pancreatic cancer remains to be one of the most challenging diseases worldwide. Pancreatic ductal adenocarcinoma (PDAC) arises in ductal epithelium of exocrine pancreas, most diagnosed only at a late stage and thereby not being resectable upon diagnosis . In the In this context, gemcitabine is a chemically nucleoside analog that is used as a first-line treatment in patients with pancreatic cancer, who previously performed a tumor resection. This strategy approach for the treatment has a reported survival of about 6 months on average . This suvia conventional pharmaceutical delivery  emissive and biodegradable polymer for fluorescent imaging of pancreatic cancers in two subcutaneous murine models. A donor unit benzodifuran(BDF)-based donor–acceptor (D–A) copolymer PBDFDTBO was synthesized  and applubstance . Moreoveubstance  or poly(ubstance  were choPEO(5000)-b-PCL(5000) and PEG(5000)-DSPE were purchased from Xi’an Ruixi Biological Technology Co., Ltd.  and Avanti Polar Lipids Inc. , respectively. PBDFDTBO (P for short in the following figures) was synthesized and characterized as previously reported . Tetrahy2O during ultrasonication with the energy of 800–900 Joule applied continuously for 1 min to form the nanoparticles. Subsequently, the mixture was transferred to the ultrafiltration tube (MWCO = 100 kDa) and centrifuged at 1500 g for 30 min to remove the organic solvent. The residues were washed three times by PBS to ensure the complete removal of the organic solvent. The obtained nanoparticles were dissolved in 200 μl of PBS and filtered using a 0.22-μm polyethersulfone filter  for sterilization purposes before in vitro or in vivo experiments.In typical synthesis, PEO-PCL or PEG-DSPE were dissolved in the THF at a concentration of 20 mg/ml, and 200 μl of the solution  was then pipetted into a glass bottle, mixed thoroughly with 10 μl of 4 mg/ml PBDFDTBO solution in the THF, before the mixture was added dropwise into 5 ml dH®PMX120) or Zetasizer Nano Analyzer . A cryogenic transmitting electron microscopy (cryo-TEM) observation of polymeric nanoparticles in solutions was carried out in a controlled-environment vitrification system. The climate chamber temperature was 25–28°C, and the relative humidity was kept close to saturation to prevent the sample evaporation during the preparation. The samples at room temperature were placed on a carbon-coated holey film supported by a copper grid and gently blotted with filter paper to obtain a thin liquid film (20–200 nm) on the grid. The grid was quenched rapidly in liquid ethane at −180°C and then transferred to liquid nitrogen (−196°C) for storage. Then, the vitrified specimen stored in liquid nitrogen was transferred to a Tecnai G2 F20 cryo-microscope, using a Gatan 626 cryoholder and its workstation. The acceleration voltage was 200 kV, and the working temperature was kept below −170°C. The images were digitally recorded with a charge-coupled device camera (Gatan) under low-dose conditions.One hundred microliters of the synthesized PEO-PCL-P or PEG-DSPE-P nanoparticles was added to a 96-well plate . First, the absorption wavelength of the two particles was measured by the microplate reader  and then the absorption peaks were determined. The corresponding wavelength under peak absorbance was selected as the excitation wavelengths to further acquire the emission spectra. The size and ζ-potential of PEO-PCL-P or PEG-DSPE-P nanoparticles were measured by Nanoparticle Tracking Analysis .Six-week-old female nude mice were subcutaneously injected with 100 μl of 5.0 × 10t-test. A p-value of <0.05 was considered to indicate a statistically significant difference.All data were shown as the mean ± standard deviation (SD). All statistical analysis was performed with GraphPad Prism 5.0 software . The statistical differences between groups were analyzed using Student’s R2 > 0.99). Due to the strong hydrophobic nature of PBDFDTBO, we adopted two PEG-based amphiphilic polymers to encapsulate this D–A copolymer inside the hydrophobic core, making it more water-soluble. The synthetic scheme was illustrated in 2O during sonication. Organic solvents were later removed, and the synthesized particles were resuspended in aqueous solutions  for the further characterizations. Absorption spectra of both synthesized PEO-PCL-P and PEG-DSPE-P nanoparticles showed the highest absorbance peak at 570 nm  is represented in t 570 nm . Both navia mouse tail vein would be diluted about 10 times by presuming the whole mouse blood volume of 2 ml). In this range, detecting the fluorescence intensity of nanoparticles represents a valid measure in evaluating the nanoparticle of an unknown concentration.To optimize the encapsulation of PBDFDTBO in the amphiphilic polymer, different initial concentrations of this D–A copolymer were employed with a fixed amount of PEO-PCL or PEG-DSPE, to prepare water-soluble nanoparticles and test their fluorescent properties in different dilutions. The results were summarized and shown in To calculate the composition of as-made nanoparticles, nanoparticles after typical synthesis were lyophilized and re-dissolved in THF, and the PBDFDTBO contained in PEO-PCL-P or PEG-DSPE-P was measured at 5.4 ± 0.3 and 7.4 ± 1.5 μg/mg nanoparticles. At the same time, the as-synthesized PEO-PCL-P or PEG-DSPE-P after typical synthesis and purification was measured at 3.6 ± 0.3 and 2.7 ± 0.2 mg, respectively; that is, 18.0 ± 1.5 mg/ml PEO-PCL-P and 13.5 ± 1.0 mg/ml PEG-DSPE-P in 200-μl aqueous solutions.2O, PBS, or serum at the same concentration as above, and tested for their photostability in different chemical environments. By normalizing each fluorescence intensity under all the tested condition (n = 5) to the highest one acquired, the results were shown in mean ± SD in 2O, PBS, and serum solutions, respectively. For PEO-PCL-P nanoparticles, the normalized intensity read was 0.98 ± 0.02, 0.98 ± 0.01, and 0.95 ± 0.02 in dH2O, PBS and serum, respectively. Therefore, the fluorescence intensity in serum dropped significantly for both nanoparticles compared with the corresponding results in water or PBS conditions (p < 0.05). Then, the synthesized nanoparticles were incubated in serum at 37°C and protected from light for 6 days, and their fluorescence intensity was measured at days 0, 2, 4, and 6, and then normalized to the highest one acquired. Results are shown in 2O, PBS, or serum were subjected to continuous photobleaching, as formulations were exposed to the excitation of 570 nm and emission intensity at 830 nm was recorded every 30 s. The fluorescence intensity was acquired by being normalized to the highest one under each condition and plotted versus time, as shown in The PEO-PCL-P or PEG-DSPE-P nanoparticles were then resuspended in dH2O and measuring their size, polydispersity index (PDI), and zeta potential over time . Results were shown in Further characterizations were investigated using TEM and NTA measurements, which corroborated the size, morphology, and surface charge of both nanoparticles . The nanp < 0.05 when compared to untreated cells), respectively. Therefore, both PEGylated nanoparticles presented a minimal toxicity to pancreatic cancer cells studied, while for the same nanoparticle, PEO-PCL-P or PEG-DSPE-P, it showed different toxicity profiles to cells with different origins.The synthesized nanoparticles were studied for their potential cytotoxicity to ensure the biosafety when applying these PEGylated nanoparticles in biological systems. Different concentrations of PEG-DSPE-P or PEO-PCL-P nanoparticles were added into the cell cultures  and tested for their toxicity at 24-h incubation. The results are shown in in vivo administration of nanoparticles, the mouse blood was obtained, and different concentrations of PEO-PCL-P or PEG-DSPE-P were added into the mouse blood for ex vivo measurement of fluorescence intensity. Results are shown in in vivo study. The nanoparticles were then intravenously administered via tail-vein injection in the subcutaneous pancreatic cancer models at a dosage of 180 mg/kg mouse for PEO-PCL-P or 135 mg/kg mouse for PEG-DSPE-P. Mouse blood was withdrawn at different time points for the pharmacokinetics study and the polymeric dye-included nanoparticles were checked by their fluorescence intensities in various organs or tumors for biodistribution and tumor accumulation studies.Mouse (PANC02) or human (8988T) PDAC cells were transplanted into the left and right flanks of nude mice, allowing for a subcutaneous tumor growth of a 2-week period. A tumor with a diameter of approximately 0.5 or 0.8 cm was formed for 8988T or PANC02 cells, respectively. Before t1/2). At 24 h p.i., the mice were sacrificed, and the major organs were extracted and analyzed by ex vivo fluorescent imaging  at a rate of 0.20 h–1 to reach the highest accumulation, that is, 5.7 ± 1.7 times the original at 24 h. Similarly, the tumor accumulation at the right flank was 14.8 ± 7.5% at a rate of 0.24 h–1 to reach the highest accumulation 6.6 ± 1.6 times the original at 24 h.For PANC02 cell-transplanted tumor models, both flanks bearing tumors were monitored by co-localizing the tumor region under the bright field with fluorescent imaging  . However, the accumulation rate and the highest accumulation between two nanoparticles in this 8988T cancer model were similar to each other.The same EO-PCL-P  or PEG-DEO-PCL-P  nanopartIn this study, a novel benzodifuran(BDF)-based D–A copolymer PBDFDTBO was synthesized and applied as a polymeric dye, which owned the emission in the NIR-I range upon excitation at 570 nm. Two differently PEGylated amphiphilic polymers, PEO-PCL and PEG-DSPE, were used to encapsulate the hydrophobic PBDFDTBO in the core to form water-soluble nanoparticles with a stable emission of NIR-I fluorescence upon excitation. The physicochemical properties of the synthesized PEO-PCL-P and PEG-DSPE-P nanoparticles were investigated, showing a close similarity to each other in many physical and chemical parameters, including their size, shape, and surface properties. For human and murine pancreatic cancer cell lines, both PEO-PCL-P and PEG-DSPE-P nanoparticles at the studied dosages exhibited a similar biocompatibility with no apparent toxicity, although the sensitivity to the cell type could be different based on the exact cell origin. In subcutaneous pancreatic cancer models of nude mice, both PEGylated nanoparticles displayed a prolonged circulation time and enhanced tumor accumulation, albeit the accumulation rates and retentions of nanoparticles in tumors were different. In fact, it was observed that PEO-PCL-P nanoparticles possessed a higher accumulation in 8988T cell-transplanted subcutaneous tumor and showed a faster accumulation in PANC02 cell-transplanted subcutaneous tumor than PEG-DSPE-P nanoparticles.in vivo but different profiles of tumor accumulation. External PEG on nanoparticles could minimize their surface energy, increasing their steric distance among nanoparticles, through the hydrogen bonds between repeating ether units and the environment solvent -block-poly(ε-caprolactone) and PEG-DSPE are among the common strategies to modify the hydrophobic substances with hydrophilic PEG on surface, to fabricate the stealth nanoparticles for enhanced blood circulation . The den solvent . In thisrounding , which, rounding . Notablyrounding . We havein vivo. Although undetermined, the interaction between PCL and PBDFDTBO favored the tumor accumulation in this study. Further experiments are ongoing to uncover the mechanism.Being hydrophobic tails, DSPE as a phospholipid owns a higher affinity to the biological membrane of bilayer than PCL, achieving a faster and easier cellular penetration and transportation . In paraCurrently, the nanomedicine research has extended to targeted molecular therapy, involving not only small-molecule chemical drugs but also a variety of biologics, including nucleotide or protein drugs and cellular immunotherapy . These tIn summary, two types of PEGylated nanoparticles were here compared to conclude an optimized coating strategy for a desired biological feature in pancreatic cancer delivery. With the same PEGylation on the outer surface, hydrophobic segments that anchor onto the encapsulated core may affect the biodistribution and tumor accumulation of the PEGylated nanoparticles, to a degree that could be determined by the hydrophobic interactions between the hydrophobic ends of amphiphilic polymers and the enwrapped substances. This study paves a new path to adjust nanoparticulate systems for enhanced permeability and retention in pancreatic tumors.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Jiangsu University Animal Ethics Administration Committee.HC, YZ, GL, and ZT conceived the idea of study. HC, YC, LX, and XZ performed the experiments. HC, YC, LX, YZ, GL, and ZT conducted the data analysis. HC, YC, LX, YZ, XZ, GL, DW, and ZT contributed to the writing of the manuscript and agreed on this submission and publication. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fuel cells are regarded as one of the most promising energy conversion devices because of their high energy density and zero emission. Development of high-performance Pt-based electrocatalysts for the oxygen reduction reaction (ORR) is vital to the commercial application of these fuel cell devices. Herein, we review the most significant breakthroughs in the development of high-performance Pt-based ORR electrocatalysts in the past decade. Novel and preferred nanostructures, including biaxially strained core–shell nanoplates, ultrafine jagged nanowires, nanocages with subnanometer-thick walls and nanoframes with three-dimensional surfaces, for excellent performance in ORR are emphasized. Important effects of strain, particle proximity, and surface morphology are fully discussed. The remaining changes and prospective research directions are also proposed. Fuel cells, as clean and effective energy conversion devices that can directly convert the chemical energy of fuels and oxidants to electricity, are highly demanded to meet the urgent requirement of vehicles and exploitation of environmentally sustainable energies . The oxipt at 0.9 V , which are 33.9 and 27.1 times greater than those of the commercial Pt/C, respectively. They also exhibited outstanding durability with negligible activity decline, unconspicuous structure, and composition changes after 50,000 cycles. The excellent ORR performance can be attributed to their unique biaxial strain, which would be beneficial to optimizing the Pt-O bond on the Pt surface and decreasing the poison effect.A class of highly uniform PtPb/Pt core/shell nanoplates was reported by Huang et al. ynthesis   nanostructures, such as nanowires, nanorods, and nanotubes, have received tremendous attention all over the world, as they may solve many of the inherent catalytic problems and enhance electrocatalytic activity toward ORR due to their uniquely anisotropic structure, which possesses high aspect ratios, fewer lattice boundaries, high electron transport characteristics, and smooth and low energy facets . Althougvia the selective dealloying approach  at 0.9 V versus the RHE is over 5 times greater than that of the commercial Pt/C catalyst (0.14 Amg-1Pt), which is attributed to the ultrathin wall thickness with a bimetallic alloyed structure. Moreover, the ORR mass activity of octahedral nanocages displayed a loss of only 36% after 10,000 cycles, which still show 3.4-fold enhancement compared with the pristine commercial Pt/C catalyst. The catalytic system here could be further optimized before it can compete with the more active system based on Pt-based alloys.PtPd nanocages with subnanometer-thick walls and controllable facets were reported by Xia et al.  etching   accessible surfaces were reported by Yang et al. via spontaneous dissolution of Ni atoms in air -like surface structure with a thickness of at least two Pt monolayers. The synergy between high specific activity and the three-dimensional open structures of Pt3Ni nanofrmes with both the internal and external accessible surfaces enabled a 22-fold improvement in mass activity compared with the commercial Pt/C. Furthermore, the Pt3Ni nanoframes showed extraordinary durability and structure stability, where the activity loss can be negligible and the frame structure remained intact after 10,000 potential cycles between 0.6 and 1.0 V. The excellent durability is attributed to the weaker oxygen binding strength from the electronic structure of the Pt-skin surface, which leads to a lower coverage of oxygenated intermediates, decreasing the probability of Pt dissolution.Highly crystalline Pts in air  , resulting in the increasing of layer spacing from 4.69 to 4.83 Å. Thus, about 3% expansion in the substrate can cause uniaxial tension on Pt nanoparticles (LCO-PtT). During discharge, the Li+ intercalated back into Li0.5CO and the lattice returned to its original spacing, generating compressive strain on Pt nanoparticles (L0.5CO-PtC)  . As a re5CO-PtC) . MoreoveEffect of particle proximity. In spite of the tremendous effort based on both experimental and computational modeling that has been dedicated to understanding the structure–performance relationship of the ORR activity on Pt-based nanostructures , it has 20 [D (cluster diameter) = 0.6 nm], Pt46 (D = 0.8 nm), and Pt>46 (D = 2.3 nm)}, were investigated  in situ characterization techniques for investigating the catalytic active sites, 2) theoretical simulations for predicting the optimized morphologies/compositions, and 3) systematic investigation on the reason of the discrepancy between liquid half-cell and full-cell tests. Besides, the development of strong Pt-C interaction will also be of great significance for achieving high-performance Pt-based ORR electrocatalysts. Deeper insights into these significant breakthroughs in the developments of Pt-based materials will help identify the optimal conditions in order to achieve Pt-based electrocatalysts with high activity and stability toward ORR. There is no doubt that the commercialization of fuel cell devices will keep moving forward as continuous and rapid development of high-performance Pt-based ORR electrocatalysts takes place.Recent years have witnessed rapid and significant progress in the synthesis of Pt-based electrocatalysts with superior ORR performance. However, the research studies in this field still face huge challenges for the practical applications of fuel cell devices, and many issues need to be addressed, such as the discrepancy between liquid half-cell and full-cell tests, the destruction of the structural integrity of these Pt-based materials, and the strategies for scale-up synthesis, including self-assembly,


The IC50 of RO4929097 was 2.93 μM. Treatment with different doses of RO4929097  effectively reduced osteoclast formation (number and resorption area) in a dose-dependent manner. The qPCR results revealed that RO4929097 attenuates RANKL-induced osteoclast formation and NFATc1 protein expression. The in vivo experiments demonstrated that RO4929097 had an inhibitory effect on LPS-induced bone resorption. Our in vitro experiments showed that RO4929097 can potently inhibit osteoclastogenesis and bone resorption by down-regulating the Notch/MAPK/JNK/Akt-mediated reduction of NFATc1. In accordance with these in vitro observations, RO4929097 attenuated LPS-induced osteolysis in mice. In conclusion, our findings indicate that Notch may represent a potential therapeutic target for the treatment of osteolytic diseases.To investigate the suppressive function of RO4929097, a potent -secretase inhibitor, on RANKL-induced osteoclastogenesis. The cytotoxicity of RO4929097 was evaluated. The suppressive effect and possible molecular mechanism of RO4929097 on RANKL-induced osteoclastogenesis was evaluated both  Bone metabolism is a dynamic process that involves the simultaneous processes of bone resorption and formation , 2 Thus,Osteoclasts (OCs) are a type of multinucleated giant cell derived from macrophage precursors. OC differentiation is the first stage of the bone resorptive process, which requires stimulation of receptor activator of NF-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF). Previous studies have shown that RANKL plays an essential role in the formation of multinucleated OCs , 9. FollIn previous studies, Notch signaling has been shown to play an essential role in multiple developmental pathways . The intin vivo results.In this study, we investigated the effect of RO4929097 on the suppression of RANKL-induced osteoclast formation and elucidated the potential molecular mechanisms associated with this process. We confirmed that RO4929097 suppressed osteoclastogenesis and bone resorption by blocking the Notch/Hes1/MAPK (Erk and p38)/Akt pathway. This conclusion was also confirmed by the BMMs were obtained and cultured as described in previous studies –21. Prim4 cells per well) in complete medium containing 33.3 ng/mL M-CSF and different concentrations of RO4929097 . Following RO4929097 treatment, the cells were incubated for different timepoints . At the end of the process, CCK-8 solution  was used according to according to the manufacturer’s guidelines.The cytotoxicity of RO4929097  was determined using an MTT assay. The BMMs were seeded into 96-well plates (1 × 104 cells per well) and stimulated with RANKL , M-CSF (33.3 ng/mL), and different concentrations of RO4929097 . The culture medium was replaced every 48 h for six days. After the osteoclasts were observed in the control group, 4% paraformaldehyde was used for OC fixation. TRAP  staining solution was added into each of the wells at 37°C for 1 h. TRAP+ cells with at least three nuclei were considered to be osteoclasts. Image J software  was used for cell enumeration.The role of RO4929097 on osteoclast formation was assessed. The BMMs were seeded into 96-well plates (1 × 105 cells/well). After 24 h, the cells were stimulated with RANKL (100 ng/mL), M-CSF (33.3 ng/mL) until OC formation was observed. The OCs (1 × 104 cells/well ) were then incubated on Corning Osteo Assay Surface plates  and cultured with M-CSF (33.3 ng/mL), RANKL (100 ng/mL), and RO4929097  for four days. A BioTek Cytation 3 Cell Imaging Reader  and Image J software were used to photograph and analyze the total resorption pits.M-CSF-dependent BMMs were incubated in six-well plates , and permeabilized for 5 min. After blocking with 1% BSA-PBS for 1 h, the cytoskeletal actin structure was stained with rhodamine-conjugated phalloidin. The immunofluorescence images were obtained using a BioTek Cytation 3 Cell Imaging Reader. Image J software was used to analyze the size (diffusion area) and number of the dental actin bands.6 cells) was extracted from the cells using TRIzol®  according to the manufacturer's protocol. A NanoDrop 2000 spectrophotometer  was used to assess the concentration of the total RNA. The following process was performed as described previously. [The total RNA  were extracted at different time points. Following treatment with 400 nM RO4929097 for 2 h, RANKL (100 ng/mL) was used to stimulate the cells for different periods of time (short time course). The membrane was blocked in 1% TBST  in 5% skim milk at room temperature for 1 h, and subsequently reacted with primary antibodies NFATc1 ; Cleaved Notch1 ; HES1 ; GAPDH ; p-Akt ; Akt ; p-ERK ; Erk ; p65 ; p-p38 ; p38 ; p-p65 . After incubating the samples overnight at 4°C, they were incubated with a secondary antibody at room temperature for 1 h using Odyssey V3 .0 image scan  to observe antibody reactivity.All processes were was carried out in compliance with the ARRIVE guidelines and under the supervision of Shanghai General Hospital Animal Center Committee of Animal Care and Use. All procedures were performed under the guidelines of the Ethical Conduct in the Care and Use of Nonhuman Animals in Research. Male C57BL/6 mice (aged 7 to 8 weeks), were randomly assigned to four groups: Control (PBS), LPS and LPS with low (400 nM) or high (800 nM) concentrations of RO4929097. A 1% pentobarbital sodium (10 mg/kg) was injected intraperitoneally to anaesthetize the mice. The knees of the mice were locally injected with 100 μL LPS or PBS (10 mg/kg), and with or without RO4929097, respectively, for two weeks. During the experiment period, no significant adverse or toxic effects were observed. The mice did not exhibit significant weight loss or lack of spirit, and none of the mice died during the experimental period. Then, euthanasia was performed using pentobarbital sodium (100 mg/kg) . The kneThe PFA-fixed mice knees were decalcified in 10% EDTA for two weeks and embedded in paraffin. Next, the samples were sliced into specific sections (4 μm thick) and subjected to H&E and TRAP staining. Digital images were obtained using an Axio ScopeA1 light microscope . The osteoclasts were enumerated using Image J software.t-test was used to assess the differences between the control and therapeutic groups. The results for the multiple group comparisons were analyzed using a Scheffe's test and one-way analysis of variance (ANOVA) with SPSS 22.0 software . Values were determined to be significant at *P < 0.05; **P < 0.01; and ***P < 0.001.All data were presented as the mean ± standard deviation (SD). A Student's The datasets supporting the conclusions of this article are available in an open access repository.The study design was carried out in compliance with the ARRIVE guidelines and approved by the Ethical Committee of Shanghai General Hospital.First, the cytotoxicity of RO4929097 was evaluated. The results showed that the IC50 of RO4929097 in the BMMs at 24 h, 48 h, and 96 h was 4.44 μM, 3.66 μM, and 2.93 μM respectively  and 1B. To assess the effect of RO4929097 on OC-mediated bone resorption activity, the resorption area on the hydroxyapatite-coated Osteo Assay plates was calculated . In accoQuantitative PCR (qPCR) was used to evaluate the relative expression of osteoclastogenesis genes at the mRNA level. It was observed that the relative gene expression of NFATc1, C-fos, Cath-K, and TRAP was markedly downregulated by RO4929097 in both a dose-dependent and time-dependent manner.  and 3B.To investigate the potential molecular mechanisms of RO4929097 in the inhibition osteoclastogenesis, the RANKL-induced NF-κB, MAPK, JNK and PI3K/Akt pathways were examined using a Western blot analysis. BMM treatment with RO4929097 during RANKL-induced osteoclast formation induced cleaved Notch1, Hes1, and NFATc1 protein expression  and 4B. in vitro experiments were used to investigate the effect of RO4929097 on RANKL-induced osteoclastogenesis and bone resorption function by studying the phenotype and associated mechanism. We investigated the function of RO4929097 in mice with LPS-induced bone resorption. According to the three-dimensional micro-CT reconstruction shown in The in vivo. In accordance with previous results, extensive osteolysis was observed in the HE-stained images of the control group, whereas a lower osteolytic level was observed in the RO4929097-treated groups /Akt signaling. In accordance with the in vitro observations, prevention of LPS-induced osteolysis in vivo also confirmed that RO4929097 may represent a potential treatment strategy for osteolytic diseases in relation to excessive osteoclast-mediated bone resorption.In conclusion, our data demonstrate that RO4929097 can effectively attenuate RANKL-induced osteoclastogenesis


Activity monitoring devices may be used to facilitate goal-setting, self-monitoring, and feedback towards a step-based physical activity (PA) goal. This study examined the performance of the wrist-worn Fitbit Charge 3™ (FC3) and sought opinions on walking and stepping-in-place from women with gestational diabetes (GDM).N = 15), with the sequence randomized. Steps were estimated by FC3 and measured, in duplicate, by direct observation . Equivalence testing by the two one-sided tests (TOST) method assessed agreement within ± 15%. Mean absolute percent error (MAPE) of steps were compared to 10%, the accuracy standard of the Consumer Technology Association (CTA)™. A subset (n = 10) completed a timed, 200-m self-paced walk to assess natural walking pace and cadence. All participants completed semi-structured interviews, which were transcribed and analyzed using descriptive and interpretive coding.Participants completed six 2-min metronome-assisted over ground bouts that varied by cadence  and mode , and gestational age 32.8 weeks (SD 2.6). The FC3 was equivalent to hand-tally for bouts of metronome-assisted walking and stepping-in-place at 84 and 100 steps per minute , although walking at 100 steps per minute (P = .01) was no longer equivalent upon adjustment for multiple comparisons . The FC3 was equivalent to hand-tally during the 200-m walk , in which mean pace was 68.2 m per minute (SD 10.7), or 2.5 miles per hour, and mean cadence 108.5 steps per minute (SD 6.5). For walking at 84 and 100 steps per minute, stepping-in-place at 100 steps per minute, and the 200-m walk, MAPE was within 10%, the accuracy standard of the CTA™. Interviews revealed motivation for PA, that stepping-in-place was an acceptable alternative to walking, and competing responsibilities made it difficult to find time for PA.Mean age was 27.0 years (SD 4.2), prepregnancy BMI 29.4 kg/mThe FC3 appears to be a valid step counter during the third trimester, particularly when walking or stepping-in-place at or close to women’s preferred cadence. Standards of Medical Care in Diabetes (Standards) recommend that after a GDM diagnosis, treatment begin with medical nutrition therapy, PA, and, depending on pregestational weight class, weight management   and comphttps://www.inqscribe.com/) software and cross-checked to ensure accuracy. A inductive thematic analysis approach was used to identify themes that emerged from the data [Audio recordings of the semi-structured interviews were transcribed using InqScribe . In regards to PA prior to pregnancy, 73.3% reported PA below recommended levels . For PA during the last 30 days, 93.3% reported PA below recommended levels . Participant characteristics are presented in Table Mean gestational age at the study visit was 32.8 weeks (SD 2.6). On average, participants were 27.0 years of age (SD 4.2) and self-reported prepregnancy BMI was 29.4 kg/mn = 10) walked at a mean cadence of 108.5 steps per minute (SD 6.5) by hand tally, and 100.7 steps per minute (SD 5.5) by the FC3 . For example, “I live in a house full of people so I get up and I’ll sweep and mop and I do laundry, do grocery shopping. I’m always out shopping so I get of lot of walking and exercise.”The semi-structured interviews revealed that participants were willing to engage in PA for health . The findings of the present study suggest that women with GDM face challenges finding time for PA given their busy schedules and competing demands. However, participants seemed open to the idea of increasing PA by walking and stepping-in-place, and to using a FC3 to record and monitor their walking and stepping. The FC3 performed adequately when walking or stepping-in-place at cadences of approximately 84 and 100 steps per minute, the latter just under their preferred walking cadence during the 200-m walk. Importantly, the FC3 met the accuracy standard of the Consumer Technology Association (CTA)™ during walking at 84 steps per minutes, walking and stepping at 100 steps per minute, and during the 200-m self-paced walk .The FC3 overestimated steps when walking or stepping at the lowest cadence examined . This is in contrast to previous studies in pregnant women, which report that devices under-estimate step counts at slower walking speeds . The 67 Previous successful lifestyle interventions for weight management  targeting pregnant and postpartum women –10 set ith week of the intervention . It is worth noting that PA decreases as pregnancy progresses [A small pilot study of a clinic-based intervention in 17 women with type 2 or gestational diabetes, who were recruited in the second trimester, included one-on-one counseling and step goal setting; counseling sessions occurred in the clinic, immediately following a prenatal care visit [ogresses , thus thThere are few published data on stepping-in-place overall. One small randomized controlled trial examined stepping-in-place or walking around the house while watching TV commercials  as convenient mode of moderate-intensity activity to replace  sedentary time . AlthougAlthough steps obtained from walking differ from those obtained while stepping-in-place, the behavioral intervention component of problem solving makes stepping-in-place attractive for this population. It can be done anywhere and simultaneously with other daily activities. However, women’s satisfaction with and enjoyment from this mode of activity, important constructs in terms of motivation for PA , remain There are other limitations worth noting. Findings from this small sample may not be generalizable, particularly the qualitative findings, given the participation rate. Finally, while consideration of MAPE is recommended because it reflects error expected at the individual level , the preResults of the present study suggest that women with GDM are willing and motivated to participate in PA for their health and that of the baby, though it needs to be simple and convenient. Walking and stepping-in-place were acceptable modes of PA, and suitable given the commonly stated barrier that it was difficult to fit PA into their busy lifestyles. Future studies are needed to understand adherence to, acceptance of and satisfaction with stepping-in-place for PA. Results also suggest that the FC3, a commercial step counter, is adequate for counting steps while walking and stepping-in-place during the third trimester of pregnancy, particularly when walking and stepping at or close to their preferred cadence.


The brain undergoes ionizing radiation exposure in many clinical situations, particularly during radiotherapy for brain tumors. The critical role of the hippocampus in the pathogenesis of radiation-induced neurocognitive dysfunction is well recognized. The goal of this study is to test the potential contribution of non-targeted effects in the detrimental response of the hippocampus to irradiation and to elucidate the mechanisms involved. C57Bl/6 mice were whole body (WBI) or partial body (PBI) irradiated with 0.1 or 2.0 Gy of X-rays or sham irradiated. PBI consisted of the exposure of the lower third of the mouse body, whilst the upper two thirds were shielded. Hippocampi were collected 15 days or 6 months post-irradiation and a multi-omics approach was adopted to assess the molecular changes in non-coding RNAs, proteins and metabolic levels, as well as histological changes in the rate of hippocampal neurogenesis. Notably, at 2.0 Gy the pattern of early molecular and histopathological changes induced in the hippocampus at 15 days following PBI were similar in quality and quantity to the effects induced by WBI, thus providing a proof of principle of the existence of out-of-target radiation response in the hippocampus of conventional mice. We detected major alterations in DAG/IP3 and TGF-β signaling pathways as well as in the expression of proteins involved in the regulation of long-term neuronal synaptic plasticity and synapse organization, coupled with defects in neural stem cells self-renewal in the hippocampal dentate gyrus. However, compared to the persistence of the WBI effects, most of the PBI effects were only transient and tended to decrease at 6 months post-irradiation, indicating important mechanistic difference. On the contrary, at low dose we identified a progressive accumulation of molecular defects that tended to manifest at later post-irradiation times. These data, indicating that both targeted and non-targeted radiation effects might contribute to the pathogenesis of hippocampal radiation-damage, have general implications for human health. The hippocampus is a highly radiosensitive brain structure involved in forming, organizing and storing memories. Its critical role in the pathogenesis of radiation-induced neurocognitive dysfunction is well recognized. Inhibition of adult hippocampal neurogenesis following whole-brain irradiation is considered one of principal mechanisms of radiation-induced cognitive dysfunction ,2. In adRadiation effects, however, are not confined to directly irradiated tissues, rather living organisms cope with injury through coordinated cell/tissue responses. Over the last 20 years, the classical nuclear target paradigm of radiation biology has been challenged by the emerging role of non-targeted effect of radiation. In fact, irradiation has been shown to affect not only the cells traversed by radiation track, but also non-irradiated neighboring cells, a response described as radiation-induced bystander effects (RIBE). Communication between irradiated and sham-irradiated neighboring cells, involving molecular signals via intercellular communication or through soluble secreted factors produced by irradiated cells, initiates RIBE and out-of-field  effects . In vitr+/−Ptch1 mice, a genetically sensitive mouse model, was increased by radiation exposure of distant tissues, indicating that there is a level of communication between irradiated and non-irradiated tissues and organs  signaling, the last one crucial for the transmission across chemical synapses A,B.p-value ≤ 0.1. We also investigated (i) the time-dependence of the modulation of miRNAs induced by exposure at 2.0 Gy in PBI and WBI hippocampi at 6 months post-irradiation and (ii) the time- and dose-dependence in PBI and WBI hippocampi exposed at 0.1 Gy at 15 days or 6 months post-irradiation. To this aim, we developed a miRNA custom PCR arrays ready to use, containing the PCR primer sets of the 25 deregulated miRNAs at 15 days after PBI irradiation with 2.0 Gy. As a criterion for deregulation, we assumed a We also analyzed whether PBI or WBI low-dose exposure with 0.1 Gy may perturb the same subset of miRNAs deregulated at 2.0 Gy. At 15 days post-irradiation C,D, we d−1), proteins  and lipids  are among the differentiating spectral features  or WBI (integrin beta-2), respectively . Four prTaken together, at 2 weeks, the proteomics data suggested a strong out-of-target effect resembling direct radiation effect in hippocampus at the 2.0 Gy but not at the 0.1 Gy dose, affecting the expression of several synaptic proteins. While the deregulation of synaptic proteins disappeared after 6 months, the proteome response in the 2.0 Gy PBI and WBI groups remained similar, now indicating changes in the level of proteins involved in catabolic activities.Adult neurogenesis is a multistep process comprising activation of quiescent NSCs, their differentiation into committed progenitor cells, neuronal survival, migration and functional integration of newborn neurons. Several factors have been shown to modulate hippocampal neurogenesis. We and others have shown that hippocampal neurogenesis in rodent models is impaired by ionizing radiation exposures ,20,21,22p = 0.0071) and a similar significant decrease of 37.70% in WBI (p = 0.0070) (p = 0.0005) and 31.65% (p = 0.0001) in PBI and WBI respectively. Finally, we observed a significant reduction of 32.77% (p = 0.046) in immature neurons labelled by doublecortin (DCX) in the DG of WBI mice but not in PBI mice, representing the unique difference in the defects induced by the two exposure modalities. Altogether, our findings clearly demonstrated that out-of-field irradiation causes defects in the dynamic transition among neural stages in the DG nearly identical to those induced by in-field irradiation 15 days after irradiation with 2.0 Gy. These defects, including self-renewal and proliferation, point to a complex disturbance in the control of NSCs progression into neurons in the hippocampus by out-of-field irradiation. However, while for WBI these defects  persisted at 6 months post-irradiation, they were transient and were fully recovered at 6 months post-irradiation for PBI exposure  B. In addexposure C.Since proteomics and miRNome analysis indicated radiation-induced modulations even at low-dose, we analyzed hippocampal neurogenesis 15 days or 6 months after irradiation with 0.1 Gy. We reported complete lack of functional deficit at 15 days post-irradiation both for WBI and PBI groups D. InsteaAbscopal effects are reported when one part of the animal’s body is exposed to radiation while another part is protected by a lead shield ,24,25,26Very little is known about the impact of abscopal effects on a shielded brain upon the irradiation of distal organs. While our pioneering work highlighted the importance of communication between irradiated and non-irradiated tissues/organs in cancer induction in vivo ,8, the pOur findings demonstrated here, for the first time, that 15 days following exposures of the lower third of the mouse body with 2.0 Gy of X-rays, the shielded hippocampus exhibited changes in miRNA and protein profiles nearly identical to those induced by WBI with the same dose. In strict concordance, the analysis of hippocampal neurogenesis at 15 days after irradiation demonstrated marked defects in the dynamic transition among neural stages, mainly involving NSCs and progenitors in the DG, nearly identical after PBI and WBI, suggesting that in-field and out-of-field irradiation induce very similar disturbance in the control of progression of NSCs into neurons in the hippocampus.Cx43 mRNA through specific binding [Cx43 deletion in which radiation-induced out-of-target tumorigenesis is drastically reduced [Notwithstanding the profuse investigations there is still much to learn about the magnitude, the molecular mechanisms and the consequences of RIBE on the brain and their contributions to the side effects of radiation therapy. Evidence suggests that non-targeted effects in non-irradiated cells may be mediated via cell-to-cell gap junctions (GJ) and through mediators released from irradiated cells, especially cytokines and chemokines ,32. Inde binding . Therefo reduced .Other miRNAs, here deregulated in response to irradiation, are involved in cellular trafficking and communication, suggesting that both PBI and WBI may target cellular trafficking in the hippocampus. Mir-1 is the top upregulated miRNA in our settings and its overexpression in the heart has been reported to attenuate hippocampal synaptic vesicle exocytosis by the posttranscriptional regulation of SNAP-25, through the transportation of exosomes . AdditioActivation of cytokines has been associated with abscopal radiation effect and macrophage activation, followed by a storm of cytokines including IL-1a, IL-1b, IL-6, TNF-α and TGF-β, accompanied the induction of abscopal radiation effects ,37,38. IAdditionally, DAG and IP3 signaling pathways are among the predicted perturbed regulatory pathways at 15 days after WBI and PBI. IP3 pathway, is involved in brain development, axonal growth, memory formation and excitability, and its deregulation contributes to the onset of many neurodegenerative diseases including Alzheimer, Amyotrophic lateral sclerosis and Autism spectrum disorders . DAG is Crucial for full neuronal functionality is the ability to modulate and adjust its proteome in response to specific cues, for example, synaptic remodeling in response to patterns of action potentials. Neuronal plasticity is the result of a balance between protein synthesis and degradation to maintain and regulate synaptic protein turnover. Significant upregulation in a subset of synaptic protein, LRRC7, SynGAP1, SHANK2, GRIN2B and SNCA, experimentally validated, pointed to important synaptic dysfunction, a prominent feature of many neuropathological conditions. Long-term proteomic changes involving synaptic plasticity have already been reported in our previous work following cranial or whole-body irradiation of in utero or neonatal mice ,47,48,49The Raman spectral data indicate the occurrence of progressive radiation-induced metabolic changes in PBI hippocampus revealed by the biochemical similarity of 2.0 Gy PBI and 2.0 Gy WBI groups at 6 months post-irradiation, while at 15 days post-irradiation the 2.0 Gy PBI group overlapped with the SI group. The CLS analysis showed significantly differentiated molecular species in the 2.0 Gy PBI and 2.0 Gy WBI groups compared to the control group including histone 2A, DNA, TNFα, TGFβ and uric acid. The proteomics analysis also found histone 2A to be significantly deregulated in the 2.0 Gy PBI and 2.0 Gy WBI groups and the miRNome analysis indicated involvement of the TGFβ signaling pathway. Cytokines, such as TNFα and TGF-β and uric acid, are considered to be danger signals released from dead or damaged cells in response to radiation ,51.Comparison of the effects at 15 days and 6 months post-irradiation allows the evaluation of the time-dependence of radiation responses after WBI and PBI with 2.0 Gy  suggestin = 1) and WBI (n = 3) with no commonly deregulated miRNAs. In addition, the number of deregulated proteins showed a strong reduction compared to the deregulation level induced at 2.0 Gy, with lack of involvement of synaptic proteins. Concordantly, analysis of neurogenesis indicated that 15 days after PBI and WBI with 0.1 Gy effects could be detected compared to the SI controls. Altogether, these data revealed only small effects at early post-irradiation times after low-dose irradiation with 0.1 Gy. Instead, the consequences of low-dose irradiation with 0.1 Gy were generally worsening with the time progressing. Biochemical profiles, for instance, indicated clear spectral differences between SI control and PBI or WBI samples at 6 months post-irradiation. In addition, the analysis of neurogenesis showed defects, although only in WBI hippocampi, consisting in a significant decrease in the pool of NSCs compartment labelled by GFAP. At 6 months post-irradiation with 0.1 Gy, we also observed an increase in the number of deregulated miRNAs that included a marked deregulation of mir-1298, controlling the GJ protein Cx43 both in PBI and WBI. Finally, we also identified miR-155 as commonly deregulated in PBI and WBI mice at 6 months after irradiation with 0.1 Gy and at both 15 days and 6 months after 2.0 Gy exposure, supporting its functional role in radiation responses. miR-155 is pro-inflammatory factor in a variety of organ systems and it is strongly upregulated following brain injuries, although whether it is beneficial [Investigation on dose-dependence of radiation responses in the hippocampus following WBI and PBI with 0.1 Gy showed the existence of a dose-response relationship. In fact, 15 days post-irradiation with 0.1 Gy a very small number of miRNAs was modulated both after PBI  operated at 250 kVp, 1 mA for 0.1 Gy and 15 mA for 2.0 Gy, with Half-Value Layer = 1.6 mm Cu . PBI was performed by exposing the lower third of the mouse body, whilst the upper two thirds were shielded with a lead-shield. Additional groups of mice were sham irradiated (SI).A dosimetric evaluation approach, based on experimental measurements with an ionization chamber NE 2571, had been employed to evaluate the dose to the shielded brain resulting from photons crossing the leads shield or deflected in the cap through the irradiated tissue. Under the adopted experimental conditions, there was no significant dose contribution to the shielded brain .http://www.r-project.org (accessed on 28 November 2018)) and the open-source Bioconductor libraries. Data were filtered based on sequence counts  and pairwise comparisons of differential miRNA expression were performed using edgeR package [p-value < 0.1 were used for gene/miRNA enrichment analysis with Cytoscape plug-in “ClueGo” (v.2.1.7) and “CluePedia” (v.1.1.7) [In this case, 15 days after irradiation, PBI, WBI and SI hippocampi were collected and total RNA was extracted using miRNeasy kit  according to the manufacturer’s instructions. Total RNA (1 μg) was converted into miRNA NGS libraries using NEBNEXT library generation kit  following manufacturer’s instructions. Samples were sequenced on the Illumina NextSeq 500 System. All sequencing data analysis was performed using the R platform  . For eacv.1.1.7) ,59.p-value ≤ 0.1.A miRNA custom PCR arrays ready to use , containing the 25 PCR primer sets of deregulated miRNAs at 15 days after irradiation with 2.0 Gy PBI, was developed. Total RNA was used as starting material for the procedure of detection and quantification of miRNA expression. Reverse transcription of total hippocampal RNA was performed with miRCURY-LNA-RT-Kit according to the manufacturer’s instructions . As a criterion for deregulation we assumed a t-test for comparison between pairs of means. p-values ≤ 0.05 were considered to be statistically significant.Fixed tissue sections were immunostained as described  using th−1 Raman line of crystalline silicon. Dark current measurement and recording of the substrate and optics signal was also performed, for data correction. As source, a 532 nm laser of ~12 mW power was focused by a 100× objective  onto the sample and the resultant Raman signals were detected using a spectrograph with a 1200 g/mm grating coupled with a CCD. Raman spectra were acquired in the 400 to 1800 cm−1 region with an integration time of 30 s per spectrum and averaged over two accumulations. Multiple calibration spectra of 1,4-Bis(2-methylstyryl)benzene were recorded along with each sample acquisition. All spectra were subsequently wavenumber calibrated using in-house developed procedures in Matlab v.9.3 . The instrument response correction was performed using the spectrum of NIST Standard Reference Material (SRM) no.2242. Spectra were recorded from the hippocampus region of the brain from 5 individual mice per group.OCT embedded brain tissue was sectioned (10 μm) using a cryostat. An XploRA confocal Raman instrument  was used for spectral acquisition. Manual calibration of the grating was carried out using the 520.7 cmAll spectral processing procedures were conducted using Matlab , along with in-house developed algorithms and procedures available within the PLS Toolbox . Briefly, spectra were imported, baseline was subtracted with a rubberband algorithm, vector normalized and smoothed using a Savitzky-Golay smoothing algorithm with a 7-point window and a 5th order polynomial.Subsequently, corrected spectra were subjected to Principal Components Analysis (PCA). In brief, PCA is a commonly used method for multivariate data reduction and visualization. It is an unsupervised method used to describe variance in data sets by identifying a new set of orthogonal features, called principal components (PCs).S, is the linear sum of contributions from spectra of pure components, a1, a2, … na, that contribute to the spectrum as follows [C1, C2,… nC, are the weights or concentrations assigned to each component spectrum. In the case of a Raman spectrum, not all contributing pure components are known. Therefore, E represents the error or residual matrix.Classical least squares (CLS) fitting analysis was performed as described previously ,61,62 us follows :S = a1C1g) and acidified with 0.5% trifluoroacetic acid (TFA) and stored at −20 °C.Each 10 µg protein sample in 0.1x RIPA buffer was subjected to tryptic digest applying a modified filter-aided sample preparation (FASP) procedure ,65. Aftem/z with automatic gain control target set to 3 × 106 and a maximum of 30 ms injection time. From the MS pre-scan, the 15 most abundant peptide ions were selected for fragmentation (MSMS) if at least doubly charged, with a dynamic exclusion of 30 s. MSMS spectra were recorded at 15,000 resolution with automatic gain control target set to 5 × 102 and a maximum of 50 ms injection time. The normalized collision energy was 28 and the spectra were recorded in profile mode.LC-MSMS analysis was performed in data-dependent acquisition (DDA) mode. MS data were acquired on a Q-Exactive HF-X mass spectrometer  each online coupled to a nano-RSLC . Tryptic peptides were automatically loaded on a C18 trap column (300 µm inner diameter (ID) × 5 mm, Acclaim PepMap100 C18, 5 µm, 100 Å, LC Packings) at 30 µL/min flow rate. For chromatography, a C18 reversed phase analytical column  at 250 nL/min flow rate in a 95 min non-linear acetonitrile gradient from 3% to 40% in 0.1% formic acid was used. The high-resolution  MS spectrum was acquired with a mass range from 300 to 1500 P-values as well as q-values based on an optimized FDR approach.Spectra were analyzed using Progenesis QI software for proteomics  for label-free quantification as previously described  with theProteome Discoverer 2.4 software  was used for peptide and protein identification via a database search (Sequest HT search engine) against SwissProt mouse data base , considering full tryptic specificity, allowing for up to two missed tryptic cleavage sites, precursor mass tolerance 10 ppm, fragment mass tolerance 0.02 Da. Carbamidomethylation of Cys was set as a static modification. Dynamic modifications included deamidation of Asn and Gln, oxidation of Met; and a combination of Met loss with acetylation on protein N-terminus. Percolator was used for validating peptide spectrum matches and peptides, accepting only the top-scoring hit for each spectrum and satisfying the cutoff values for FDR < 1%, and posterior error probability <0.01. The final list of proteins complied with the strict parsimony principle.t-test approach described in [https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis [The quantification of proteins was based on abundance values based on the area for unique plus razor peptides. Abundance values were normalized in a retention time dependent manner to account for sample loading errors. The protein abundances were calculated summing up the abundance values for admissible peptides. The final protein ratio was calculated using median abundance values of 4 to 5 replicate analyses each and defined to be up to 100. The statistical significance of the ratio change was ascertained employing the ribed in  which isanalysis , accesseThe immunoblotting was performed as described previously . The folFor proteomics, the following filtering criteria were used: proteins identified and quantified with at least two unique peptides (UP), had a q-value of ≤0.05 and fold-changes of ≤0.77 or ≥1.3 were considered as significantly differentially expressed. Six biological replicates per treatment group were included in the analysis.t test. The error bars represent standard error of the mean (±SEM) . Each treatment group was compared individually to the sham-irradiated control group.For immunoblotting, statistical analysis was performed with Graph Pad prism software  using an unpaired Student’s The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner In summary, we successfully identified molecular alterations in non-coding RNAs, proteins and metabolic levels as well as histological changes in the rate of hippocampal neurogenesis following PBI and WBI, clearly demonstrating the existence of non-targeted radiation effects in mouse hippocampus. Predicted pathway analysis identified alterations in DAG/IP3 and TGF-β signaling pathways as well as changes in the expression level of proteins involved in the regulation of long-term neuronal synaptic plasticity and synapse organization, coupled with defects in NSCs self-renewal in the hippocampus. At moderate doses (2.0 Gy) the majority of the PBI effects were transient, well detectable at early post-irradiation times and decreasing at 6 months post-irradiation, indicating mechanistic difference with the long-lasting WBI effects. The opposite was observed at low dose (0.1 Gy), with a progressive accumulation of cellular and molecular defects becoming more manifested at 6 months post-irradiation.Even though our mechanistic observations are referred to a mouse model, our conclusions, emphasizing that both targeted and non-targeted radiation effects play a role in the pathogenesis of hippocampal radiation-damage, might have more general implications for human health. For instance, region-sparing during radiotherapy, aimed at dose reduction to regions of neurogenesis, may be less effective than expected due to of out-of-target effects coming from other irradiated tissue/organs. Elucidating the pathogenic biological and molecular mechanism underlying out-of-target radiation effects in the brain is relevant for prevention of radiation-induced brain injury and optimization of therapeutic strategies for the treatment of damage stemming from radiotherapy.


Certification of seed potato as free of viruses is essential for stable potato production. Among more than 30 virus species infecting potato, potato leafroll virus (PLRV), potato virus S (PVS), potato virus X (PVX), and potato virus Y (PVY) predominate worldwide and should be the targets of a high-throughput detection protocol for seed potato quarantine.EF1α). Virus-specific primers were derived from conserved regions among randomly selected representatives considering viral genomic diversity. Our assay simultaneously detected representative Japanese isolates of PLRV, O lineage of PVS, PVX, and NTN strain of PVY. The variability of melting temperature (Tm) values for each virus was confirmed using Japanese isolates, and virus species could be identified by the values of 87.6 for PLRV, 85.9 for PVX, 82.2 (Ordinary lineage) to 83.1 (Andean lineage) for PVS, and 79.4 (NA-N strain) to 80.5 (O strain and NTN strain) for PVY on average. The reliability of calculation was validated by comparing the calculated Tm values and measured Tm values and the values had a strong linear correlation (correlation of determination: R2 = 0.9875). Based on the calculated Tm values, representative non-Japanese isolates could also be identified by our assay. For removing false positives, two criteria were set for the evaluation of result; successful amplification was considered as 30.0 ≥ threshold cycle value, and the virus-specific peak higher than the EF1α-specific peak was considered as positive. According to these criteria, our assay could detect PLRV and PVS from 100-fold dilution of potato leaf homogenate and PVX and PVY from 1000-fold in a model assay.We developed an assay based on one-step real-time multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with melt curve analysis for the four viruses and one internal control, potato elongation factor 1 alpha gene (This new high-throughput detection protocol using one-step real-time mRT-PCR was sensitive enough to detect viruses in a 100-fold dilution of singly-virus contaminated homogenate in a model assay. This protocol can detect the four viruses in one assay and yield faster results for a vast number of samples, and greatly save the labor for seed potato quarantine and field surveys.The online version contains supplementary material available at 10.1186/s12985-021-01591-3. Solanum tuberosum L.), the fourth most important crop in the world [Potato , Andean lineage (PVSA), and Phureja lineage (PVSP) [As a high-throughput alternative, one-step real-time mRT-PCR is a desirable candidate. The real-time PCR technique is classified into two methods, one method using fluorescent-labeling probes and the other using double-stranded DNA (dsDNA) binding fluorescent dye. The latter, the intercalator method, is more cost-effective because only two fluorescent dyes, a dsDNA binding fluorescent dye and a reference dye for each well’s standardized fluorescent intensity, need to be compared with fluorescent-labeling probes to each target in the former method. In the intercalator method, the DNA dye is captured to dsDNA, not to single-stranded DNA (ssDNA), in a target non-specific manner, and amplicons can be identified by the melting temperature (Tm), which is measured as the release of the dye during denaturing dsDNA to ssDNA . Among intercalator methods, only one assay, which can identify multiple amplicons of potato viruses with different Tm values by the melt curve analysis, was reported . Their ae (PVSP) . PVY is e (PVSP) . In addie (PVSP) , so theiHere, we developed a new one-step real-time mRT-PCR assay for the four major potato viruses and used a plant gene as the internal control. Combined with the simple RNA preparation method , the detAll viral isolates in the previous report were also used in this study . The virhttps://www.ncbi.nlm.nih.gov/), considering lineages of PVS and strains of PVY. Alignment analysis was performed using the MEGA-X program (https://www.megasoftwa re.net/) to determine the conserved regions. For internal control, which was an indicator of successful RNA preparation and DNA amplification, five sequences of mRNA for potato elongation factor 1 alpha gene (EF1α)  were used and the conserved region was also determined using MEGA-X. The melting temperature (Tm) value of primer was calculated by Oligo calculator , and all primers were designed to be around 60 °C in their Tm values, except degenerated primers for PVS which were resulted in the range of 60–65 °C. The Tm values for predicted amplicons were calculated by uMelt batch software of DNA-UTAH.ORG (https://dna-utah.org/index.html) with the following options: 30 mM mono cation, 1.2 mM free Mg2+, 10% dimethyl sulfoxide (DMSO), “Huguet et al. [Primers for PLRV, PVS, PVX, PVY were designed based on conserved regions among more than six representative sequences  were used. Primer concentrations were optimized by comparing the heights of each specific peak in the melt curve for the four viruses contaminated samples to adjust the amplification efficiency for simultaneous detection of all four viruses. All primer concentrations were initially set at 0.2 µM in the reaction mixture and decreased or increased depending on the height of peaks. For simultaneous detection of the four viruses, the RNA solution was prepared from a potato leaf homogenate containing all four viruses, prepared by mixing an equal volume of four potato leaf homogenates, each containing one of the four viruses.For this experiment, isolates ChLR_2 for PLRV, M for PVSPCR Procedure was as follows. The RNA solution was extracted using a simple filter paper-based method . The RNAR2) for the regression line, which was given by the spreadsheet program, Microsoft Excel 365. The calculation of Tm values for non-Japanese isolates was performed as described above.A non-specific peak was observed in the melt curve for virus-free potato leaf and similar to the PVS-specific peak in the multiple detections. The assay was performed to detect three viruses without PVS genome or PVS primers to reveal whether the peak in multiple detections was a specific or non-specific product. For the assay without PVS genome, RNA solutions were prepared from each singly-virus-infected leaf homogenate, except PVS-infected, and an equal volume of the three solutions was mixed and used as a template. For the assay without PVS primers, the template solution was prepared from the mixture of the four potato leaf homogenates. To validate the calculation, calculated Tm values for Japanese isolates in Table O, O-IC249 for PVX, and Eu-12Jp for PVYNTN. The detection sensitivity was confirmed using tenfold dilution series for each virus, prepared by mixing with singly-virus-infected and virus-free potato leaf homogenate. The RNA preparation, the assay, and the analysis of the result were done as described above.The identical four virus isolates were used in this experiment: ChLR_2 for PLRV, M for PVSA and PVSP , O strain (PVYO), O5 strain (PVYO5), Eu-N strain (PVYEu-N), NA-N strain (PVYNA-N) and E strain (PVYE) [Considering genetic diversity of PVS and PVY, primer sequences reported by Cheng et al.  were comn (PVYE) . The seqEF1α , and the calculated Tm value seemed to be sufficiently reliable to presume Tm values for non-Japanese isolates using their genome sequences. Based on the calculation, Tm values for representatives were not overlapped with other viruses, and the values for PVY were from 77.5 to 79.5 °C, PVS from 81.0 to 82.5 °C, PVX from 84.5 to 85.5 °C, and PLRV from 87.0 to 87.5 °C in ascending order  to 83.1 °C (PVSA) and from 79.4 (PVYNA-N) to 80.5 °C (PVYO or PVYNTN), respectively. This result was consistent with the previous study on real-time mRT-PCR assay with melt curve analysis for four grapevine viruses, and the values for each virus species were also varied about 1.0 °C according to isolates in their report [O and isolate Leona of PVSO. The actual Tm values for these isolates in our assay were unknown, and the Tm values for these isolates might be more closed to each other than our calculation. The shape of the peak for PVY in the melt curve was sharper than that for PVS; this feature would be useful for diagnosing these two viruses. Our primers would be able to identify representatives by Tm values and the shape of the peak.There was no real-time RT-PCR assay with melt curve analysis which would detect all Japanese isolates of the four viruses. All primers for the four potato viruses were newly designed in the conserved region among representatives not to overlap amplicon Tm values with the other species to meet our requirement. Amplicon Tm values for PVS and PVY were within a broad range and varied by lineages of PVS and strains of PVY, from 82.2 (PVSr report . We coulEF1α, so we would suggest the below criterion removed the non-specific peak. The Ct value for RNase-free water as the negative control was over 30.0, so the criterion for successful amplification was also set. Namely, these two criteria were set for our assay.Successful amplification is “Ct value ≤ 30.0.”Virus-specific peak higher than the internal-control peak is evaluated as positiveOur assay could detect the four viruses simultaneously from the mixture of four homogenates of singly-virus-infected potato leaves using newly designed primers. A few real-time mRT-PCR assays with melt curve analysis for plant viruses were reported , 17–19, EF1α-specific primers do not cross exon and detect both mRNA and genomic DNA for EF1α. The peak for EF1α in melt curve analysis will somehow overestimate if crude RNA solution is applied as a template. The result showed that the specific peaks for all four viruses were observed in 1000-fold dilution. However, the 1000 dilution of PVS and PLRV were evaluated as negative by the second criterion set above. Our assay with criteria could detect any of four viruses from 100-fold dilution and seemed to have enough sensitivity to detect each virus in Japanese seed potato quarantine.The two criteria also affect the evaluation of detection sensitivity. The detection sensitivity of real-time RT-PCR assays has been confirmed using purified genome RNA  and plasIn Japan, plants are grown out from tubers, and the leaves of the plants are used for viral detection because the virion of PVY, which is the most common virus in the world, is gradually degrading during the dormancy period of tubers until breaking the dormancy . Our orgOur labor-saving high-throughput detection assay for internationally important potato viruses PLRV, PVS, PVX, and PVY can be used with the simple RNA preparation method we reported previously. This protocol is useful for detecting viruses in many samples for seed potato quarantine and field surveys.Additional file 1. Supplementary Figure 1. The primer binding position in viral genome. The position referred to the representative sequences written in Virus Taxonomy ninth edition: GenBank accession X14600 for PLRV, AJ863509 for PVS, D00344 for PVX, and AJ890348for PVY. Primer binding sequences and their direction were shown under each alignment, and red letters meant the position of mismatches between primer and genome sequences, and degenerated nucleotides were replaced with represented nucleotides. Abbreviations for lineages of PVS; O, PVSO; A, PVSA; P, PVSP, and those for strains of PVY; C, PVYC; NA-N, PVYNA-N; E, PVYE; O5, PVYO5; Eu- N, PVYEu-N; O, PVYO. Supplementary Figure 2. The confirmation of mismatches between viral genome and reported primers. The position referred to the representative sequences written in Virus Taxonomy ninth edition: GenBank accession AJ863509 for PVS and AJ890348 for PVY. Pimers’ binding sequences and their direction were shown under each alignment, and red letters meant the position of mismatches between primer and genome sequences and degenerated nucleotide was replaced with represented nucleotide. Abbreviations for lineages of PVS; O, PVSO; A, PVSA; P, PVSP, and those for strains of PVY; C, PVYC; NA-N, PVYNA-N; E, PVYE; O5, PVYO5; Eu-N, PVYEu- N; O, PVYO.


Acinetobacter baumannii. Carbapenems are the principal agents used to treat A. baumannii infections. However, when strains develop resistance to this class of antibiotics, colistin is considered one of the last-resort drugs. However, the appearance of resistance to colistin also makes treatment of the Acinetobacter infections very difficult. Antimicrobial peptides (AMP) from the innate immunity hold promise as new alternative antibiotics due to their multiple biological properties. In this study, we characterized the activity and the membrane-perturbing mechanism of bactericidal action of a derivative of a frog-skin AMP, namely Esc(1-21), when used alone or in combination with colistin against multidrug-resistant A. baumannii clinical isolates. We found that the mixture of the two compounds had a synergistic effect in inhibiting the growth and killing of all of the tested strains. When combined at dosages below the minimal inhibitory concentration, the two drugs were also able to slow down the microbial growth and to potentiate the membrane-perturbing effect. To the best of our knowledge, this is the first report showing a synergistic effect between AMPs, i.e., Esc(1-21), and colistin against colistin-resistant A. baumannii clinical isolates, highlighting the potential clinical application of such combinational therapy.Multidrug-resistant microbial infections and the scarce availability of new antibiotics capable of eradicating them are posing a serious problem to global health security. Among the microorganisms that easily acquire resistance to antibiotics and that are the etiological cause of severe infections, there is  Acinetobacter baumannii is a Gram-negative bacterium involved in the etiology of a wide range of clinical manifestations, including hospital-acquired pneumonia, urinary tract infections, and bloodstream and surgical site infections, which occur preferentially in patients admitted to intensive care units  [A. baumannii has been invoked as an independent risk factor of mortality [A. baumannii is still open. Of paramount importance in this debate is the observation that a huge number of clinically isolated strains of A. baumannii are MDR [Acinetobacter spp. belong to four phylogenetic subgroups: OXA-23-like; OXA-24-like; OXA-51-like and OXA-58. OXA-51-like subgroups are intrinsic to A. baumannii, occurring in most or all of the strains [A. baumannii infection often requires the use of colistin, available commercially as colistin sulfate for oral and topical use, and as colistimethate sodium for parenteral and aerosol therapy [A. baumannii infections. From this scenario, it is evident that the identification of new therapeutic strategies, as well as the elaboration of new classes of molecules able to counteract A. baumannii infections, is highly pressing. Given the lack of effective drugs treatment and the high cost of new antibiotic monotherapies, antibiotics combination provides a strong weapon to defeat antibiotic resistance [Pseudomonas aeruginosa and to enhance the growth inhibition activity of aztreonam [A. baumannii strains along with the underlying molecular mechanism. Infections sustained by multi-drug-resistant bacteria (MDR), i.e., bacteria resistant to at least three different classes of antimicrobial agents, represent a serious threat for human health. ections) . The colortality , though  are MDR ; carbape are MDR . However are MDR . Class D strains . The pan strains  include  therapy , colisti therapy , thus cosistance ,13,14,15sistance ,17,18,19sistance ,22,23,24ztreonam ,26. In tA. baumannii were isolated from different clinical samples. We initially evaluated their susceptibility to colistin and to Esc(1-21), by the broth microdilution assay to determine the minimal inhibitory concentration (MIC). As indicated in Four strains  of blaOXA-51 gene, confirming the taxonomic assignment as A. baumannii, and blaOXA-23 gene. The three isolates #1, #3, and #4 were assigned to sequence group (SG) 1 and associated with international clones (ICL) II, as they amplified genes of length equal to 580 bp (csuE), 162 bp (blaOXA-51), and 343 bp (ompA); only the #2 strain belonged to the SG4, which has not yet been related to any clonal lineage. All isolates showed the intrinsic FIC) were lower than the corresponding MICs when tested alone, giving a FIC value ranging from 0.25 to 0.37 for all of the selected strains. This proves the occurrence of a strong synergism between the two compounds in inhibiting the growth of all A. baumannii strains.Subsequently, the checkerboard assay was used to identify the possible existence of a synergistic antimicrobial activity by the combination of the two antimicrobials in comparison to the individual activities. As reported in FIC, there was no increase in optical density for strains #1, #3, and #4 within 24 h indicating a complete inhibition of bacterial growth, while for strain #2 a slight increase of optical density was recorded above 18 h. The bacterial growth kinetics of the four strains are shown in FIC in combination with 1/2, 1/4 and 1/8 MICFIC of the antibiotic. Indeed, as reported in The antimicrobial efficacy of colistin-Esc(1-21) mixture was also observed when Esc(1-21) was used at its MICFIC were withdrawn, 24 h after incubation at 37 °C, and seeded on agar plates for colonies counting. Interestingly, no viable bacterial cells were obtained for #1, #3, and #4 strains indicating that combination of the two compounds at their MICFIC had a complete bactericidal activity. In comparison, this effect was less pronounced for strain #2, with a killing activity of ~99%.To further investigate whether the antimicrobial activity of the drugs combination was accompanied by a bactericidal effect, aliquots from samples containing the two drugs at their MICA. baumannii species and to characterize the kinetics of membrane perturbation, the membrane-impermeable fluorescent probe Sytox Green was employed. Its fluorescence intensity significantly increases upon binding to nucleic acids once it has entered cells with a damaged membrane. As shown in A. baumannii strains, a fast and dose-dependent membrane perturbation process was induced by Esc(1-21). This is manifested by the rapid increase of fluorescence intensity within the first minutes of peptide addition  is the perturbation of the cytoplasmic membrane. To verify whether this mechanism was preserved against FIC, was added to concentrations of colistin ≤MIC i.e., 4, 2, 1 and 0.5 mg/L (dotted lines), the kinetics of membrane perturbation became faster with a similar profile to that of the frog-skin AMP at the corresponding concentration .The exact mechanism of action of the cationic colistin is still unclear, but the primary microbicidal effect is believed to be triggered by its electrostatic interaction with the negatively-charged lipid A of lipopolysaccharides (LPS), the major components of the outer membrane in Gram-negative bacteria. When the permeabilization of the outer membrane occurs, colistin translocates through it in a self-promoted manner, and subsequently provokes the destabilization of the inner membrane with consequent cell death. As reported in A. baumannii strains, but unfortunately resistance to it has frequently been described [pmrCAB locus which regulates the phosphoethanolamine (pEtN) transferase with replacement of phosphate groups of lipid A by phosphoethanolamine; (ii) impairment of LPS synthesis by ramA locus mutations; (iii) activation of efflux pumps such as KpnEF, AcrAB, and Sap proteins systems [A. baumannii is nowadays considered one of the most relevant clinical microorganisms, being the etiological agent of a large variety of nosocomial infections, encompassing bacteremia, meningitis, pneumonia, and urinary tract infections [blaOXA-like) promoted carbapenem resistance in A. baumannii isolates. Currently, OXA-58 oxacillinases have drastically decreased in Italy, being replaced by OXA-23 [A. baumannii strains analyzed in our work, all phenotypically resistant to carbapenem as well as to colistin (MIC > 4 mg/L), were consistent with the MDR A. baumannii strains circulating in Italy in the last decade, carrying the blaOXA-23 carbapenemase gene: three isolates  were related to ICL II, while #2 strain belonged to SG4, which is not related to any ICL. Due to multiple resistance mechanisms, involving β-lactamases production, enhanced expression of efflux pumps, alteration of target sites of conventional antibiotics, and decreased membrane permeability, A. baumannii causes infections that make even last-line antibiotics or antibiotic-combination therapies almost ineffective.As a polymyxin antibiotic, colistin is a cationic polypeptide that exerts its antibacterial effect against a wide range of Gram-negative bacteria. Although the exact mechanism of bacterial killing of polymyxins is not clearly defined, colistin affects the permeability of the LPS-outer membrane through the electrostatic interaction with the negatively-charged lipid A . Colistiescribed . Colisti systems ,29. A. bfections ,30,31. TA. baumannii. As an example, Jangra and coworkers [A. baumannii strains and obtained FIC values ranging from 0.25 to 0.5 [A. baumannii used in their study, except for one strain against which a synergistic effect was observed (FIC = 0.5) [A. baumannii. However, this strain had only an intermediate resistance to colistin (MIC = 1 mg/L) [A. baumannii (MIC of colistin > 4 mg/L). We previously discovered an additive effect of Esc(1-21) in inhibiting the growth of the Gram-negative bacterium Pseudomonas aeruginosa when combined with aztreonam and hypothesized that Esc(1-21) served as helper agent to facilitate the intracellular influx of the antibiotic, allowing it to exert the toxic effect on bacteria, by blocking peptidoglycan crosslinking [A. baumannii, despite the fact that this effect was more limited versus the SG4 strain. AMPs constitute a valid alternative against multidrug-resistant bacteria either if used alone or in combination with traditional antibiotics . In the oworkers  tested t5 to 0.5 . Jahangi5 to 0.5 , proved C = 0.5) . Lately,C = 0.5)  showed a 1 mg/L) . To the slinking . In the A. baumannii [Klebsiella pneumoniae [A. baumannii cells, our data have emphasized the possibility to re-evaluate the last resort antibiotic colistin, thus opening new avenues based on a combinatorial AMP/antibiotic-based therapy for treatment of MDR A. baumannii infections resistant to colistin.Although AMPs have been found to be active against colistin-resistant aumannii ,40,41, seumoniae . OverallThe AMP Esc(1-21) was purchased from Biomatik . It was assembled by stepwise solid-phase synthesis and purified via reverse-phase high-performance liquid chromatography to a purity of 98% using a gradient of acetonitrile in 0.1% aqueous trifluoroacetic acid (from 28% to 100% in 30 min) at a flow rate of 1.0 mL/min. The molecular mass was verified by electron spray ionization mass spectrometry. Colistin sulfate was purchased from Sigma-Aldrich .A. baumannii (n = 4) used in the study were isolated from four patients admitted to the Intensive Care Unit of a 1300-bed tertiary care academic hospital . The strains #1 and #4 were isolated from blood cultures, while the strains #2 and #3 were isolated from respiratory secretions. The samples were cultured on agar media  incubated for 24 h at 37 °C. The bacterial colonies were identified by MALDI-TOF mass spectrophotometry , with a discriminatory score > 2300 [The strains of e > 2300 .blaOXA-like). The sequences of blaOXA-like alleles encoding carbapenemases were aligned and group-specific regions were identified using Bioedit software [A. baumannii. These primers were combined with six new primers that were designed to amplify fragments of genes encoding acquired OXA-23-like (501 bp: 5′-GAT CGG ATT GGA GAA CCA GA and 5′-ATT TCT GAC CGC ATT TCC AT), OXA-24-like (246 bp: 5′-GGT TAG TTG GCC CCC TTA AA and 5′-AGT TGA GCG AAA AGG GGA TT) and OXA-58-like (599 bp: 5′-AAG TAT TGG GGC TTG TGC TG and 5′-CCC CTC TGC GCT CTA CAT AC), OXA-143-like (149 bp: 5′-TGG CAC TTT CAG CAG TTC CT and 5′-TAA TCT TGA GGG GGC CAA CC) carbapenemases (Since carbapenem-resistant isolates are producing oxacillinases enzymes belonging to molecular class D (OXA enzymes), multiplex PCR was performed to identify resistance genes belonging to oxacillinases  was used for rapid assignment of A. baumannii isolates into three major PCR-based groups (Gs) corresponding to international clones I (G2), II (G1), and III (G3) [ompA, csuE, and blaOXA-51-like sequence groups defined as Group 1 and Group 2 were performed using the primers listed in A typing scheme based on two multiplex PCRs targeting three genes under selective pressure (III (G3) . MultiplompA and csuE were designed from sequences available at GenBank  or from sequences determined during initial studies (DQ289014–DQ289019). These were aligned and consensus primers were designed from common sequence areas. The amplification conditions were: 94 °C for 3 min, followed by 30 cycles of 94 °C for 45 s, 57 °C for 45 s, and 72 °C for 1 min, followed by a final extension at 72 °C for 5 min [ompA fragment in the Group 2 PCR, and the amplification of only the csuE and blaOXA-51-like fragments in the Group 1 PCR.Primers for or 5 min ,48. Iden5 CFU/mL) in the wells of a sterile round bottom polystyrene microplate  and incubated at 37 °C for 24 h. The latest EUCAST interpretative criteria of the MICs for colistin were used, considering as resistant the strains with an MIC ≥ 4 mg/L [Antimicrobial susceptibility was performed by broth microdilution assay using Microscan Walkaway System  following the manufacturer’s instructions. The minimal inhibitory concentration (MIC) for colistin and Esc(1-21) was assessed by serial twofold dilutions in cation-adjusted Mueller-Hinton II broth (CAMHB) in a manual broth microdilution method, carried out by inoculating 100 µL of a bacterial suspension . The interaction of colistin (A) with Esc(1-21) peptide (B) was evaluated by the fractional inhibitory concentration index (ΣFIC). This index was calculated according to the equation: ΣFIC = FICA + FICB =  + . According to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines, the combination was considered synergistic when the ΣFIC is <0.5, indifferent when the ΣFIC is ≥0.5 to <2, and antagonistic when the ΣFIC is ≥2. The checkerboard titration method was used for the assessment of the synergistic activity of colistin in combination with Esc(1-21), following the CLSI guidelines ,51. BrieA. baumannii strains in the presence of colistin and Esc(1-21) scalar concentrations was also tested kinetically in sterile microplates using a microtiter plate reader  as previously described for the checkerboard assay. The sample absorbance was automatically detected at OD590 at two-hour intervals for 24 h at 37 °C. The results were used to construct the bacterial growth curves, correlating to the absorbance in function of time. In addition, to evaluate a possible killing effect, aliquots of 30 µL, drawn from the FIC wells, were seeded on Mueller-Hinton II agar plates  and incubated at 37 °C for 24 h.The growth dynamics of the four A. baumannii when used alone or in combination, the Sytox Green assay was performed as previously reported [7 CFU/mL were incubated with 1 μM Sytox Green in PBS for 5 min in the dark. After peptide addition, changes in fluorescence intensity  caused by the binding of the dye to intracellular DNA were monitored for 120 min in a microplate reader  at 37 °C. Controls were cells not treated with the peptides.To assess the ability of Esc(1-21) and colistin to perturb the cytoplasmic membrane permeability of reported . ApproxiA. baumannii clinical isolates, probably through the potentiation of the membrane-perturbing activity of the compounds. The potential clinical application of such combination therapy should be further evaluated.Our data clearly showed that the combination of the AMP Esc(1-21) and colistin have a synergistic effect in inhibiting the growth and in killing colistin-resistant